Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Controllable genome editing with split-engineered base editors

Nature Chemical Biology volume 17, pages 1262–1270 (2021)Cite this article

Subjects

Abstract

DNA deaminase enzymes play key roles in immunity and have recently been harnessed for their biotechnological applications. In base editors (BEs), the combination of DNA deaminase mutator activity with CRISPR–Cas localization confers the powerful ability to directly convert one target DNA base into another. While efforts have been made to improve targeting efficiency and precision, all BEs so far use a constitutively active DNA deaminase. The absence of regulatory control over promiscuous deaminase activity remains a major limitation to accessing the widespread potential of BEs. Here, we reveal sites that permit splitting of DNA cytosine deaminases into two inactive fragments, whose reapproximation reconstitutes activity. These findings allow for the development of split-engineered BEs (seBEs), which newly enable small-molecule control over targeted mutator activity. We show that the seBE strategy facilitates robust regulated editing with BE scaffolds containing diverse deaminases, offering a generalizable solution for temporally controlling precision genome editing.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Split deaminases show activity in vitro and in cells.
Fig. 2: Split-engineered BEs represent a generalizable strategy to enable small-molecule-controlled editing.
Fig. 3: Split-engineered BEs permit efficient editing across genomic sites.
Fig. 4: Split-engineered BEs decrease off-target effects.
Fig. 5: seBEs permit temporal control over base editing.

Data availability

High-throughput RNA-seq. data are deposited at the Gene Expression Omnibus database (accession number GSE181109). Individual amplicon sequencing data are available as Supplementary Information and raw reads will be available upon request. Novel plasmids used in this study are available from Addgene: AID’-BE4max (Addgene no. 174696), CMV-AID’-seBE4max-IRES (Addgene no. 174697), CMV-A3A-seBE4max-IRES (Addgene no. 174698), CMV-evoA1-seBE4max-IRES (Addgene no. 174699), Lenti-evoA1-BE4max (Addgene no. 174700), Lenti-evoA1-seBEn_empty (Addgene no. 174701) and Lenti-evoA1-seBEc (Addgene no. 174702). Nucleic acid sequences of all constructs used in this study are provided in a note at the end of the Supplementary Information. Source data are provided with this paper.

References

  1. Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).

    Article  CAS  PubMed  Google Scholar 

  2. Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci. Adv. 3, eaao4774 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Liu, L. D. et al. Intrinsic nucleotide preference of diversifying base editors guides antibody ex vivo affinity maturation. Cell. Rep. 25, 884–892.e3 (2018).

    Article  CAS  PubMed  Google Scholar 

  8. Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods 13, 1029–1035 (2016).

    Article  CAS  PubMed  Google Scholar 

  9. Green, A. M. & Weitzman, M. D. The spectrum of APOBEC3 activity: from anti-viral agents to anti-cancer opportunities. DNA Repair 83, 102700 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Feng, Y., Seija, N., D I Noia, J. M. & Martin, A. AID in antibody diversification: there and back again. Trends Immunol. 41, 586–600 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Siriwardena, S. U., Chen, K. & Bhagwat, A. S. Functions and malfunctions of mammalian DNA-cytosine deaminases. Chem. Rev. 116, 12688–12710 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Burns, M. B. et al. APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494, 366–370 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Robbiani, D. F. & Nussenzweig, M. C. Chromosome translocation, B cell lymphoma, and activation-induced cytidine deaminase. Annu. Rev. Pathol. 8, 79–103 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Roberts, S. A. et al. An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers. Nat. Genet. 45, 970–976 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kim, D. et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat. Biotechnol. 35, 475–480 (2017).

    Article  CAS  PubMed  Google Scholar 

  16. Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275–278 (2019).

    Article  CAS  PubMed  Google Scholar 

  18. Kim, D., Kim, D. E., Lee, G., Cho, S. I. & Kim, J. S. Genome-wide target specificity of CRISPR RNA-guided adenine base editors. Nat. Biotechnol. 37, 430–435 (2019).

    Article  CAS  PubMed  Google Scholar 

  19. Grunewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).

    Article  CAS  PubMed  Google Scholar 

  21. Grunewald, J. et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat. Biotechnol. 37, 1041–1048 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Rees, H. A., Wilson, C., Doman, J. L. & Liu, D. R. Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci. Adv. 5, eaax5717 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Nuñez, J. K., Harrington, L. B. & Doudna, J. A. Chemical and biophysical modulation of Cas9 for tunable genome engineering. ACS Chem. Biol. 11, 681–688 (2016).

    Article  PubMed  Google Scholar 

  24. Lim, S. A. & Wells, J. A. Split enzymes: design principles and strategy. Methods Enzymol. 644, 275–296 (2020).

    Article  CAS  PubMed  Google Scholar 

  25. Gangopadhyay, S. A. et al. Precision control of CRISPR-Cas9 using small molecules and light. Biochemistry 58, 234–244 (2019).

    Article  CAS  PubMed  Google Scholar 

  26. Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).

    Article  CAS  PubMed  Google Scholar 

  27. Iyer, L. M., Zhang, D., Rogozin, I. B. & Aravind, L. Evolution of the deaminase fold and multiple origins of eukaryotic editing and mutagenic nucleic acid deaminases from bacterial toxin systems. Nucleic Acids Res. 39, 9473–9497 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ear, P. H. & Michnick, S. W. A general life-death selection strategy for dissecting protein functions. Nat. Methods 6, 813–816 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Mok, B. Y. et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 583, 631–637 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Qiao, Q. et al. AID recognizes structured DNA for class switch recombination. Mol. Cell 67, 361–373.e4 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Gajula, K. S. et al. High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase. Nucleic Acids Res. 42, 9964–9975 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wang, M., Yang, Z., Rada, C. & Neuberger, M. S. AID upmutants isolated using a high-throughput screen highlight the immunity/cancer balance limiting DNA deaminase activity. Nat. Struct. Mol. Biol. 16, 769–776 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Cabantous, S., Terwilliger, T. C. & Waldo, G. S. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat. Biotechnol. 23, 102–107 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Kohli, R. M. et al. A portable hotspot recognition loop transfers sequence preferences from APOBEC family members to activation-induced cytidine deaminase. J. Biol. Chem. 284, 22898–22904 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wang, M., Rada, C. & Neuberger, M. S. A high-throughput assay for DNA deaminases. Methods Mol. Biol. 718, 171–184 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36, 950–953 (2018).

    Article  CAS  Google Scholar 

  37. Gehrke, J. M. et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Landry, S., Narvaiza, I., Linfesty, D. C. & Weitzman, M. D. APOBEC3A can activate the DNA damage response and cause cell-cycle arrest. EMBO Rep. 12, 444–450 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Voß, S., Klewer, L. & Wu, Y. W. Chemically induced dimerization: reversible and spatiotemporal control of protein function in cells. Curr. Opin. Chem. Biol. 28, 194–201 (2015).

    Article  PubMed  Google Scholar 

  40. Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Thuronyi, B. W. et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat. Biotechnol. 37, 1070–1079 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Mak, C. H., Pham, P., Afif, S. A. & Goodman, M. F. A mathematical model for scanning and catalysis on single-stranded DNA, illustrated with activation-induced deoxycytidine deaminase. J. Biol. Chem. 288, 29786–29795 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    Article  CAS  PubMed  Google Scholar 

  44. Doman, J. L., Raguram, A., Newby, G. A. & Liu, D. R. Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat. Biotechnol. 38, 620–628 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Jin, S. et al. Rationally designed APOBEC3B cytosine base editors with improved specificity. Mol. Cell 79, 728–740.e6 (2020).

    Article  CAS  PubMed  Google Scholar 

  46. Wang, L. et al. Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations. Nat. Cell Biol. 23, 552–563 (2021).

    Article  PubMed  Google Scholar 

  47. Chen, H. et al. The mTOR inhibitor rapamycin suppresses DNA double-strand break repair. Radiat. Res. 175, 214–224 (2011).

    Article  CAS  PubMed  Google Scholar 

  48. Pearce, S. & Tucker, C. L. Dual systems for enhancing control of protein activity through induced dimerization approaches. Adv. Biol. 5, e2000234 (2021).

    Article  Google Scholar 

  49. Wang, X. et al. Cas12a base editors induce efficient and specific editing with low DNA damage response. Cell. Rep. 31, 107723 (2020).

    Article  CAS  PubMed  Google Scholar 

  50. Tsai, S. Q. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Schutsky, E. K. et al. Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase. Nat. Biotechnol. 36, 1083–1090 (2018).

    Article  CAS  Google Scholar 

  52. Schutsky, E. K., Nabel, C. S., Davis, A. K. F., DeNizio, J. E. & Kohli, R. M. APOBEC3A efficiently deaminates methylated, but not TET-oxidized, cytosine bases in DNA. Nucleic Acids Res. 45, 7655–7665 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Mol, C. D. et al. Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA. Cell 82, 701–708 (1995).

    Article  CAS  PubMed  Google Scholar 

  54. Lee, M. et al. Engineered Split-TET2 enzyme for inducible epigenetic remodeling. J. Am. Chem. Soc. 139, 4659–4662 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Xu, Y. et al. A TFIID-SAGA perturbation that targets MYB and suppresses acute myeloid leukemia. Cancer. Cell. 33, 13–28.e8 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36, 888–893 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Tarumoto, Y. et al. LKB1, salt-Inducible kinases, and MEF2C are linked dependencies in acute myeloid leukemia. Mol. Cell 69, 1017–1027.e6 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Shi, J. et al. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nat. Biotechnol. 33, 661–667 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Matsuda, T. & Cepko, C. L. Controlled expression of transgenes introduced by in vivo electroporation. Proc. Natl Acad. Sci. USA 104, 1027–1032 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to M. Weitzman and K. Musunuru for helpful discussions. This work was in part supported by the Penn Center for Genomic Integrity and the US National Institutes of Health (NIH) through grant nos. R01-GM138908 and R01-HG010646 (to R.M.K.). K.N.B is an NSF Graduate Research Fellow. N.H.E. was supported by NIH T32-GM007170 and F30-HG011578. A.M.G. was supported by grant no. NIH K08-CA212299.

Author information

Authors and Affiliations

  1. Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Kiara N. Berríos & Tong Wang

  2. Graduate Group in Cell and Molecular Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Niklaus H. Evitt

  3. Department of Pediatrics, Washington University School of Medicine, St. Louis, MI, USA

    Rachel A. DeWeerd & Abby M. Green

  4. Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Diqiu Ren & Junwei Shi

  5. Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Meiqi Luo, Aleksia Barka & Rahul M. Kohli

  6. Epigenetics Institute, Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Caroline R. Bartman, Yemin Lan, Junwei Shi & Rahul M. Kohli

  7. Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Caroline R. Bartman & Junwei Shi

  8. Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Rahul M. Kohli

Authors
  1. Kiara N. Berríos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Niklaus H. Evitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rachel A. DeWeerd
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Diqiu Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Meiqi Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Aleksia Barka
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Caroline R. Bartman
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yemin Lan
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Abby M. Green
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Junwei Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Rahul M. Kohli
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.N.B., J.S. and R.M.K. conceived the approach and designed the research. K.N.B., N.H.E., R.A.D., D.R., M.L., A.B., T.W., C.R.B. and A.M.G. performed experiments and analyzed data. K.N.B., N.H.E., T.W. and Y.L. performed computational and statistical analysis. The manuscript was drafted by K.N.B., revised by N.H.E., J.S. and R.M.K., with added input and approval from all authors.

Corresponding authors

Correspondence to Junwei Shi or Rahul M. Kohli.

Ethics declarations

Competing interests

K.N.B., J.S. and R.M.K. through the University of Pennsylvania have filed a patent application on aspects of this work. R.M.K. is on the Scientific Advisory Board for Life Edit, Inc.

Additional information

Peer review information Nature Chemical Biology thanks Reuben Harris, Moritoshi Sato and other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Intact, inserted, and split DNA deaminase constructs with AID*.

(a) Construct schematics for AID* and AID*-INS variants (analogous to those in Fig. 1b) that were used to determine the impact of optGFP insertion in E. coli. Numbers above the constructs represent the amino acid sequence of the deaminase itself. (b) Construct schematics for co-expression or individual expression of AID*-SPL2N and AID*-SPL2C fragments in E. coli. (c) Left—an in vitro assay to measure deaminase activity on a 3’-fluorescein (FAM) labeled oligonucleotide substrate. UDG, uracil DNA glycosylase. Right—a representative denaturing gel (100 nM DNA, variable enzyme concentration) is shown, along with substrate and product controls (C and U, respectively). Analysis of product formation with three independent replicates was used to define the dose-response curve provided in Fig. 1d.

Source data

Extended Data Fig. 2 Intact, inserted, and split DNA deaminase constructs with A3A.

(a) Construct schematics for A3A and A3A-INS2 variants used to determine the impact of optGFP insertion in E. coli. Numbers above the constructs represent the amino acids of the deaminase itself. (b) Left—an in vitro assay to measure deaminase activity on a 3’-fluorescein (FAM) labeled oligonucleotide substrate. UDG, uracil DNA glycosylase. AP, abasic site. Middle—a representative denaturing gel (100 nM DNA, variable enzyme concentration) is shown, along with unreacted substrate and product controls (C and U, respectively). Right—product formation was quantified as a function of enzyme concentration (n = 3) and fit to a sigmoidal dose-response curve to determine the amount of enzyme needed to convert half of the substrate (EC50) under these fixed reaction conditions with 95% confidence interval shown. Each data point represents the mean and standard deviation across three biological replicates. (c) Construct schematics for mammalian expression of A3A-INS2, A3A(E72A)-INS2, and A3A-SPL2 variants used to determine the impact of optGFP insertion on the DNA damage response in HEK293T cells (d) HEK293T cells were transfected with catalytically inactive mutant A3A(E72A)-INS2, A3A-INS2, or co-transfected with A3A-SPL2N and A3A-SPL2C. After transfection, cells were stained for γH2AX and sorted for both GFP and γH2AX expression. The bar plot depicts frequency of GFP+ or GFP+/γH2AX+ cells after transfection of HEK293T cells with the indicated constructs, corresponding to the representative histogram shown in Fig. 1e. The mean, standard deviation, and individual observations from independent biological replicated are shown. (e) Representative immunofluorescent images of transfected U2OS cells are shown, corresponding to Fig. 1e. DAPI stain highlights the nucleus, GFP staining shows insert expression or split reconstitution, and γH2AX serves as a marker of active A3A-mediated DNA damage. A 10 µm scale bar is provided. Representative experiments were repeated independently three times and the results were reproducible.

Source data

Extended Data Fig. 3 Intact and split-engineered base editor constructs.

(a) Parent construct schematics for intact BE4max scaffold editors with AID’, evoA1, and A3A. (b) Construct schematics for split-engineered seBE-T2A editors with AID’, evoA1, and A3A. Constructs were created by insertion of a cassette that splits the intact deaminase into two fragments, separated by a self-cleaving T2A peptide. At the bottom is the alternative seBE construct strategy, where the T2A fragment is replaced with an internal ribosome entry sequence (IRES) that leads to expression of two independently translated split protein fragments (denoted as seBEN and seBEC) with no need for protease processing.

Extended Data Fig. 4 Split-engineered base editors with A3A enable small-molecule-controlled editing.

Editing efficiency is evaluated in a d2gfp-HEK293T cell line. The presence of d2gfp-targeting sgRNA can introduce a stop codon (Q158*) and abrogate fluorescence to generate GFPoff cells, which can be tracked by either flow cytometry or deep-sequencing. Two-sided Mann–Whitney test was performed on all comparisons between means in this figure (n.s., not significant; *p ≤ 0.05, exact p-values provided as statistical source data files). (a) Representative flow cytometry histograms associated with transfection of intact or seBE-T2A constructs with or without rapamycin. (b) Mean, standard deviation and individual data points shown for quantification of GFPoff cells by flow cytometry (c) Left—deep sequencing results demonstrating C to T conversion efficiency at the target cytosine. The mean, standard deviation and three biological replicates are shown. Fold-change (FC) is the ratio of mean values for the higher versus lower condition. Right—editing footprints across the d2gfp locus for A3A-BE4max and A3A-seBE-T2A in the absence or presence of rapamycin. The numbers mark the distance from the protospacer adjacent motif site. The target cytosine base is noted with a blue arrow. Data represent position-wise averages of three biological replicates, with individual replicate data provided in Supplementary Table 1. (d) sgRNA-independent off-target genomic editing in cells transfected with intact A3A-BE4 or A3A-seBE-IRES and sgRNA targeting EXM1, along with dSaCas9-sgRNA targeting a different locus. Left—EMX1 on-target editing. Right—off-target editing at the locus opened by dSaCas9. The mean, standard deviation, and three biological replicates are shown. Editing footprints for each locus and replicates are provided in Supplementary Fig. 4. (e) Quantification of γH2AX upon base editor expression with or without rapamycin. HEK293T cells were transfected with an empty vector (pcDNA-EV), catalytically inactive mutant A3A(E72A)-BE4max, A3A-seBE-T2A, A3A-seBE-IRES, intact A3A-BE4max, or isolated A3A domain (pcDNA-A3A) and an EMX1-targeting sgRNA expressing GFP. Cells were either maintained with or without rapamycin and then stained for γH2AX. Shown are the percentage of γH2AX positive cells in the GFP positive population. The mean, standard deviation, and three biological replicates are shown. individual observations are shown (n = 3). Two-sided Mann-Whitney test was performed (n.s., not significant; *p ≤ 0.05, exact p-values provided as statistical source data files).

Source data

Extended Data Fig. 5 Base editing efficiency in human cells at diverse loci and off-target sites.

HEK293T cells were untreated or transfected with intact BE4max or seBE constructs in the absence or presence of rapamycin. C or G describes whether the coding (C) or non-coding (G) strand cytosine is targeted, respectively, with the subscript denoting the position of the sgRNA targeted by the base editor. Shown are experiments with control constructs and (a) evoA1-seBE-T2A (corresponding to Fig. 3b and Fig. 4b), (b) evoA1-seBE-IRES (corresponding to Fig. 3c and Fig. 4b), and (c) AID’-seBE-IRES (corresponding to Fig. 3c and Fig. 4b). Bars indicate mean and error bars indicate standard deviations of n = 3 biological replicates. Complete editing footprints for each locus and replicate in (a–c) are provided in Supplementary Figs. 1–3. (d) Western blot of cells transfected with evoA1-based constructs in the absence and presence of rapamycin. The Cas9 antibody probes the intact editor and the seBEC fragment (which are of similar size); Hsp90 antibody serves as a loading control; c-myc antibody probe the N-terminal tag of the seBEN fragment. Representative experiment was repeated independently two times and the results were reproducible.

Source data

Extended Data Fig. 6 Split engineered base editors show low transcriptome-wide C to U mutations.

Total RNA was analyzed using the RADAR pipeline (RNA-editing Analysis-pipeline to Decode All twelve-types of RNA-editing events). The unique edits for each sample were cataloged by removing edits contained in either of the sgRNA-only samples. Left—shown the pie charts indicating the type of edit in each of the three independent replicates. Right—mean fractions of specific edits across the three replicates are provided.

Extended Data Fig. 7 Lentiviral constructs for intact and split-engineered base editor editing in K562 cells.

(a) Lentiviral construct schematics for intact BE4 max, sgRNA-only, seBEC and seBEN + sgRNA are shown. (b) Quantification of GFPoff cells by flow cytometry for cells with EMX1-targeting sgRNA along with intact BE4max or seBE-IRES. Cells were either treated with no rapamycin or with rapamycin (25 nM) added at either day 3 or day 5 (marked with arrow) and then maintained continuously. Mean and standard deviation are noted, with individual data points shown (n = 3).

Extended Data Fig. 8 Gating strategies for flow cytometry plots.

For all samples, viable cells were selected based on a forward scatter vs side scatter plot. Viable cells were then analyzed by the gating strategies shown. (a) Gating strategy for intact, inserted, and split A3A constructs shown in Fig. 1e and Extended Data Fig. 2d. (b) Gating strategy for base editing constructs generating GFPOFF cells shown in Fig. 2c and Extended Data Fig. 4a. Left - After sorting for live cells, a second gate for efficiently transfected cells was applied. Right – the GFPOFF population was then quantified. (c) Gating strategy for AID’- and A3A-based BE4max and seBE4max constructs shown in Fig. 4d and Extended Data Fig. 4e.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4 and Note.

Reporting Summary

Supplementary Table 1

Individual replicates of d2GFP amplicon sequencing data.

Supplementary Table 2

Unique RNA C>U edits data table.

Supplementary Table 3

Cloning and sequencing primers.

Source data

Source Data Fig. 1

Unprocessed gels from Fig. 1d.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed gels from Extended Data Fig. 1c.

Source Data Extended Data Fig. 2

Unprocessed gels from Extended Data Fig. 2b.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Unprocessed western blots from Extended Data Fig. 5d.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Berríos, K.N., Evitt, N.H., DeWeerd, R.A. et al. Controllable genome editing with split-engineered base editors. Nat Chem Biol 17, 1262–1270 (2021). https://doi.org/10.1038/s41589-021-00880-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-021-00880-w

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing